Methods for the detection of analytes in small-volume blood samples

ABSTRACT

This invention is in the field of medical devices. Specifically, the present invention provides portable medical devices that allow detection of analytes from a biological fluid. The methods and devices are particularly useful for providing point-of-care testing for a variety of medical applications.

CROSS REFERENCE

This application is a continuation application of U.S. Ser. No.11/939,509, filed on Nov. 13, 2007, which claims the benefit of U.S.Provisional Application No. 60/865,805 filed Nov. 14, 2006, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Many medical procedures require tests to be performed with a sample of apatient's fluid. The ability to rapidly and accurately detect a widerange of analytes present in a bodily fluid is often critical fordiagnosis, prognosis, and treatment of diseases.

Traditionally, detecting a range of analytes present in a bodily fluidsuch as blood has been performed in laboratories by trained technicians.Performing such assays is usually time-consuming and costly. The desirefor rapid turnaround time creates a need to facilitate testing that canbe delivered at the point-of-care. Point-of-care testing is particularlydesirable because it rapidly delivers results to medical practitioners,enables faster consultation, and avoids unattended deterioration of apatient's condition.

Although several point of care testing devices are available, themajority of which is adapted to detect a single analyte, or one type ofanalytes for a single indication. Examples of such point of care devicesare tests for glucose, drugs of abuse, serum cholesterol, pregnancy, orovulation.

Thus, there remains a need for alternative designs of point of caresystems that are capable of detecting a range of analytes from bodilyfluid. A desirable system would allow quantitative and qualitativemeasurements of analytes in a more cost effective and timely fashion.The present invention addresses this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

One aspect of the present invention is the design of a system to effectdetection of different analytes in a bodily fluid. In one embodiment,the present invention provides a system that typically comprises a) afluidic device comprising a cartridge, said cartridge comprising asample collection unit and an assay assembly, wherein said samplecollection unit allows a sample of bodily fluid to react with reactantscontained within said assay assembly to yield a colored product havingan absorbance spectrum corresponding to at least one wavelength from alight source; b) a light source transmitting the at least one wavelengthto the assay assembly; and c) a detector that detects absorption oflight of the at least one wavelength, wherein said absorption isindicative of the presence of the analyte in said bodily fluid. Ingeneral, the amount of absorption is related to the concentration of theanalyte in the bodily fluid. Preferably, the amount of absorption isstoichiometrically related to the concentration of the analyte in thebodily fluid. The subject system is preferably configured to be apoint-of-care system.

In a related but separate embodiment, the present invention provides afluidic device capable of detecting the presence or absence of ananalyte in a bodily fluid from a subject. The fluidic device can be partof the system described above. The subject fluidic device typicallycomprises (a) a cartridge, said cartridge comprising a sample collectionunit, an assay assembly, and (b) a light source, wherein said samplecollection unit is configured to collect a sample of bodily fluid fromsaid subject and wherein said assay assembly comprises at least onereaction site containing a reactant that reacts with said analyte toyield a colored product having an absorbance spectrum corresponding toat least one wavelength from said light source. Where desired, thefluidic device can be employed to detect a plurality of analytes.

The assay assembly employed in the subject fluidic device or system isgenerally configured to run an enzymatic assay yielding a coloredproduct. The assay assembly can be configured to run assays capable ofdetecting a wide variety of analytes. Non-limiting exemplary analytesinclude drug, drug metabolite, biomarker indicative of a disease, tissuespecific marker, and tissue specific enzyme. Preferred analytes fordetection include without limitation HDL cholesterol, LDL cholesterol,total cholesterol, lipids, and glucose. Where desired, the assayassembly is configured to run an immunoassay.

The light source employed in the subject fluidic device or systemtypically produces at least one wavelength corresponding to theabsorbance spectrum of the colored product generated by an assay. Asuitable light source can comprise a light emitting diode and/orluminescent paint. Where luminescent paint is used as the light source,it is typically coated on the assay assembly.

The present invention also provides a method of detecting an analyte ina bodily fluid from a subject. The method typically involves the stepsof a) introducing a sample of bodily fluid into a fluidic devicecomprising a sample collection unit and an assay assembly, said assayassembly comprising reactants that are capable of reacting with saidanalytes; b) allowing said sample of bodily fluid to react with saidreactants contained within said assay assembly to yield a coloredproduct having an absorbance spectrum corresponding to at least onewavelength from a light source; c) transmitting the at least onewavelength to the fluidic device from said light source; and d)detecting absorption of light of the at least one wavelength transmittedto the fluidic device, wherein said absorption is indicative of thepresence of the analyte in said bodily fluid. The method can be employedto detect analytes in a sample of bodily fluid that is less than about500 ul, less than about 50 ul, or less than about 20 ul, or even lessthan about 10 ul. Where desired, the methods can be applied to detectanalytes in a predetermined amount of bodily fluid that can beundiluted, unprocessed or diluted or processed by, e.g., filtration,centrifugation and other like processes.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts an exemplary point-of-care system of the presentinvention.

FIG. 2 shows a perspective view of various layers of an exemplaryfluidic device of the present invention.

FIGS. 3 and 4 illustrate the fluidic network within an exemplary fluidicdevice.

FIG. 5 illustrates an exemplary sample collection unit of the presentinvention.

FIG. 6 illustrates an exemplary sample collection well in fluidiccommunication with a metering channel, and a metering element.

FIG. 7 shows an exemplary fluidic network between a metering channel, amixing chamber and a filter.

FIG. 8 shows an absorption spectrum of a Trinder product.

FIG. 9 shows the spectral response of an assay simulation using a lightsource having an emission spectrum that perfectly overlaps with anabsorption spectrum of an absorbing species.

FIG. 10 shows a light attenuation response for a cholesterol assay.

FIG. 11 shows simulation of the signal modulation for an assay in whicha light emission spectrum and an absorption spectrum of the absorbingspecies overlaps, and the λ max varies between the two spectrums by 50nm.

FIG. 12 shows simulation of the signal modulation for an assay in whicha light emission spectrum and an absorption spectrum of the absorbingspecies overlaps, and the λ max varies between the two spectrums by 50nm, where an edge filter with a cut-off below the lower λ max is used oneither the emission or detection side of the optical system.

DETAILED DESCRIPTION OF THE INVENTION

System and Fluidic Device

One aspect of the present invention is a system for detecting an analytein a sample of bodily fluid from a subject. The terms “subject” and“patient” are used interchangeably herein, which refer to a vertebrate,preferably a mammal, more preferably a human. Mammals include, but arenot limited to, murines, simians, humans, farm animals, sport animals,and pets.

The system is capable of detecting and/or quantifying analytes that areassociated with specific biological processes, physiological conditions,disorders or stages of disorders.

The subject system typically comprises a fluidic device having one ormore of the following components: a sample collection unit, an assayassembly, a light source, a detector, and optionally a communicationassembly. In one embodiment, the subject system comprises: a) a fluidicdevice comprising a cartridge, said cartridge comprising a samplecollection unit and an assay assembly, wherein said sample collectionunit allows a sample of bodily fluid to react with reactants containedwithin said assay assembly to yield a colored product having anabsorbance spectrum corresponding to at least one wavelength from alight source; b) a light source transmitting the at least one wavelengthto the assay assembly; and c) a detector that detects absorption oflight of the at least one wavelength, wherein said absorption isindicative of the presence of the analyte in said bodily fluid.

Sample Collection Unit:

The sample collection unit typically allows a sample of bodily fluid tobe collected from a subject to react with reactants contained within theassay assembly for generating a signal indicative of the presence of theanalyte of interest. The sample collection unit may take a variety ofconfigurations so long as it collects and delivers the sample of bodilyfluid to the assay assembly. In some embodiments, the sample collectionunit is in fluidic communication with one or more components of thesubject system or fluidic device.

Where desired, the sample collection unit is configured to collect asample of bodily fluid from the subject and to deliver a predeterminedportion of the sample to be assayed by the assay assembly. In thismanner, the device automatically meters the appropriate volume of thesample that is to be assayed. The sample collection unit can comprise asample collection well, a metering channel, and a metering element.Generally, the sample collection well collects the bodily fluid from thepatient. The metering channel is in fluidic communication with thesample collection well and is dimensioned to collect the predeterminedportion of the sample to be assayed. The metering element is adapted toprevent a volume of sample larger than the predetermined portion of thesample from being assayed.

FIG. 5 illustrates a top view of an exemplary sample collection unit(SCU) showing sample collection well (SCW) in fluidic communication withmetering channel (MC), and metering element (ME).

As shown, the sample collection well (SCW) comprises a through hole witha larger diameter at the top tapering to a smaller diameter at thebottom. The through hole is intended to be the location where the sampleis provided to the fluidic device, such as by fingerstick or pipettedblood. The sample collection well (SCW) may be any inlet which allowsfor a sample to be received by the fluidic device.

The metering channel (ME) can be in fluidic communication with thesample collection well (SCW) to receive the sample. The metering channel(MC) has a proximal end (PE) and a distal end (DE). The distal end (DE)of the metering channel (MC) can include a stop junction (SJ) as will bedescribed below.

In some illustrative embodiments the metering channel (MC) is about 10mm long and has a cross section of about 1 mm². In other embodiments themetering channel (MC) is about 12.5 mm long and is about 0.9 mm wide andabout 0.9 mm high.

A predetermined portion of sample as used herein can generally refer tothe volume of sample inside the metering channel (MC) between the stopjunction (SJ) and the metering element (ME) after it has closed thefluidic connection between the sample collection well (SCW) and themetering channel (MC). In some embodiments the dimensions of themetering channel (MC) typically determines the volume of thepredetermined portion of sample. The volume of a predetermined portionin a subject sample collection unit (SCU) may be less than 50, less than40, less than 30 or 20 microliters. In a preferred embodiment, thevolume of a predetermined portion is about 10 microliters.

The metering channel (MC) is preferably capable of holding, prior toactuation of the metering element (ME), a volume of sample greater thanthe predetermined portion such that the stop junction (SJ) does notallow sample to flow into the mixing chamber (MiC) when stressed by ahydrostatic pressure of sample from the sample collection unit (SCU).

In some embodiments the metering element is adapted to prevent a volumeof sample greater than the predetermined portion from being assayed.Generally, the metering element (ME) can be adapted to pinch off thesample inside the metering channel (MC) from the sample collection well(SCW). The metering element (ME) can be a one-time valve initially openand adapted to be actuated by mechanical action by the reader assembly,as described herein. FIG. 6 is a perspective view of the meteringelement (ME) as a pin shown in an open, or unactuated, position that canbe mechanically actuated by the reader assembly to close off the fluidicconnection between the sample collection well (SCW) and the meteringchannel (MC). The metering element (ME) can take any shape and can be ofany size, and can be moved into a position to prevent a volume of samplegreater than the predetermined portion from being assayed by anytechnique, e.g., manual force or magnetic force.

In some embodiments the metering channel (MC) has a stop junction (SJ)at its distal end (DE). In FIG. 5, stop junction (SJ) comprises meteringchannel (MC) opening into the larger mixing chamber (MiC), therebycreating an abrupt end to the capillary dimensions of metering channel(MC). The stop junction (SJ) is shown comprising a right-angled junctionbetween the metering channel (MC) and the mixing chamber (MiC).

The stop junction (SJ) can be adapted to prevent sample from flowinginto the mixing chamber (MiC) before the predetermined portion of samplehas been metered. While the stop junction (SJ) as shown in FIG. 5 doesnot comprise any moveable elements, the stop junction (SJ) may alsocomprise a valve or other blocking element that prevents thepredetermined portion of sample from flowing from the metering channel(MC) into the mixing chamber (MiC).

An alternative method of loading the sample into the fluidic device isby side loading rather than loading the sample onto the top of thefluidic device. In such an embodiment, the metering channel (MC)terminates on the side or preferably, at a corner, of the cartridge. Themetering channel (MC) can be in direct communication with the mixingchamber (MiC) and the diluent chamber (DC) can be connected by a channelto the metering channel (MC) similar to the top loading embodimentabove. The sample can be drawn into the metering channel (MC) bycapillary action but does not enter the diluent flush channel (DFC) asthat channel is initially sealed from the metering channel (MC). Theuser or an automated mechanism in the reader assembly then seals theproximal end (PE) of the sample capillary prior to actuating thedilution operation as described above.

In some embodiments the inner surface of the sample collection well(SCW) and/or the metering channel (MC) may be coated with a surfactantand/or an anti-coagulant solution. The surfactant provides a wettingsurface to the hydrophobic layers of the fluidic device and facilitatefilling of the metering channel (MC) with the fluid sample, e.g., blood,such that the wetness of the metering channel (MC) can not be so largethat the stop junction (SJ) cannot contain the blood at the distal end(DE) of the metering channel (MC). The anti-coagulant solution can helpprevent the sample, e.g., blood, from clotting when provided to thefluidic device. Exemplary surfactants that can be used include withoutlimitation, Tween, Triton, Pluronic and other non-hemolytic detergentsthat provide the proper wetting characteristics of a surfactant. EDTA isa non-limiting anti-coagulant that can be used.

In one embodiment the solution comprises 2% Tween, 25 mg/mL EDTA in 50%Methanol/50% H₂O, which is then air dried. A methanol/water mixtureprovides a means of dissolving the EDTA and Tween, and also driesquickly from the surface of the plastic. The solution can be applied tothe layers of the fluidic device by any means that will ensure an evenfilm over the surfaces to be coated, such as, e.g., pipetting, spraying,or wicking.

In some embodiments the sample collection unit (SCU) also comprises adilution chamber (DC) in fluidic communication with the metering channel(MC), wherein the dilution chamber (DC) is configured to store a diluentand comprises a port for engaging pressure means for transferring thediluent from the dilution chamber (DC) into the metering channel (MC).FIG. 5 shows dilution chamber (DC) and diluent flush channel (DFC)fluidly connecting dilution chamber (DC) with the metering channel (MC).The diluent flush channel (DFC) can be adapted to be filled with diluentfrom the dilution chamber (DC).

In some embodiments the sample collection unit (SCU) further comprises amixing chamber (MiC) in fluidic communication with the metering channel(MC), wherein the mixing chamber (MiC) is configured to mix thepredetermined portion of the sample with the diluent to yield a dilutedsample. An exemplary mixing chamber (MiC) is shown in FIG. 5. The mixingchamber (MiC) is preferably dimensioned such that the intersectionbetween the metering channel (MC) and the mixing chamber (MiC) creates astop junction (SJ) to prevent the predetermined portion of sample fromentering the mixing chamber (MiC) until the diluent flushes the sampleinto the mixing chamber (MiC).

In some embodiments the mixing chamber (MiC) includes a movable mixingelement (MME) that causes the mixing of the predetermined portion of thesample with the diluent. Exemplary moveable mixing element (MME) isshown in FIG. 5 with a general ball shape.

In one embodiment the movable mixing element (MME) is magneticallycontrolled, e.g., a magnetically controlled ball in the mixing chamber(MiC) that, when magnetically controlled, will cause the mixing of thepredetermined portion of the sample and the diluent. The ball can beabout 5% of the combined volume of the sample and diluent. The ball canbe magnetically controlled to move in a reciprocal, linear fashion,within the mixing chamber (MiC).

The moveable mixing element (MME) is shown inside the mixing chamber(MiC), however, it is contemplated that the mixing element may operateoutside of the fluidic device, for example when the reader assembly isadapted to agitate the fluidic device and thereby mixing thepredetermined portion of sample and the diluent.

In some embodiments the sample collection unit (SCU) further comprises afilter (F) configured to filter the diluted sample before it is assayed.Exemplary filter (F) is shown in FIG. 5. In some embodiments the filter(F) is fluidly connected to and downstream to the mixing chamber (MiC)as shown in FIG. 5.

While the sample collection unit (SCU) can include a dilution chamber(DC), mixing chamber (MiC), and a filter (F), it is contemplated thatsome or all of these components may not be included in the samplecollection unit (SCU). It may, for example, be unnecessary to filter asample and thus the sample collection unit (SCU) may not have a filter.

FIG. 7 shows an exemplary fluidic network between a metering channel, amixing chamber and a filter.

In some embodiments it may be desirable to detect the presence ofanalytes on a cell surface, within a cell membrane, or inside a cell.The difficulty of detecting such analytes is that cells and other formedelements are particulate and components of cells do not readily interactwith traditional assay chemistries which are designed to operate onanalytes in solution. Cell-surface analytes react slowly andinefficiently with surface bound probes, and analytes inside the cellcan not react at all with bound probes. To allow the detection of suchanalytes, in some embodiments the fluidic device may include a lysingassembly to lyse cells present in the bodily fluid sample. The lysingassembly may be incorporated with the sample collection unit, a dilutionchamber, and/or a filtration chamber. In some embodiments the samplecollection unit, dilution chamber, and lysing component are within thesame element in the fluidic device. In some embodiments the lysingcomponent may be incorporated with an assay reagent described below.

Where desired, lysing agents may be impregnated and then dried intoporous mats, glass fiber mats, sintered frits or particles such asPorex, paper, or other similar material. Lysing agents may be dried ontoflat surfaces. Lysing agents may also be dissolved in liquid diluents orother liquid reagents. In some embodiments porous materials are used tostore the lysing agents because they can store a lysing agent in dryform likely to be very stable. They can also facilitate the mixing ofthe bodily fluid sample with the lysing agent by providing a tortuouspath for the sample as it moves through the porous material. In someembodiments such porous materials have a disc shape with a diametergreater than its thickness. In some embodiments lysing agents may bedried onto porous materials using lyophilization, passive evaporation,exposure to warm dry flowing gas, or other known methods.

A variety of lysing agents are available in the art and are suitable foruse in connection with the subject fluidic device. Preferred lysingagents are non-denaturing, such as non-denaturing detergents.Non-limiting examples of non-denaturing detergents include thesit,sodium deoxylate, triton X-100, and tween-20. The agents are preferablynon-volatile in embodiments where the agents are impregnated into asolid porous materials. In some embodiments lysing agents are mixedtogether. Other materials may be mixed with the lysing agents to modifythe lytic effects. Such exemplary materials may be, without limitation,buffers, salts, and proteins. In some embodiments lysing agents will beused in amounts that are in excess of the minimum amount required tolyse cells. In some embodiments lysing agents will be used that can lyseboth white and red cells.

The sample collection unit can be adapted to receive any bodily fluidssuspected to contain an analyte of interest, such bodily fluids includebut are not limited to blood, serum, saliva, urine, gastric anddigestive fluid, tears, stool, semen, vaginal fluid, interstitial fluidsderived from tumorous tissue, and cerebrospinal fluid.

The volume of bodily fluid to be received in the sample collection unitis generally less than about 500 microliters, or may be less than about50 microliters.

In some embodiments, the bodily fluids are used directly for detectingthe analytes present therein with the subject fluidic device withoutfurther processing. Where desired, however, the bodily fluids can bepre-treated before performing the analysis with the subject fluidicdevices using any methods described herein or known in the art. Thechoice of pre-treatments will depend on the type of bodily fluid usedand/or the nature of the analyte under investigation. For instance,where the analyte is present at low level in a sample of bodily fluid,the sample can be concentrated via any conventional means to enrich theanalyte. Methods of concentrating an analyte include but are not limitedto drying, evaporation, centrifugation, sedimentation, precipitation,and amplification. Where the analyte is a nucleic acid, it can beextracted using various lytic enzymes or chemical solutions according tothe procedures set forth in Sambrook et al. (“Molecular Cloning: ALaboratory Manual”), or using nucleic acid binding resins following theaccompanying instructions provided by manufactures. Where the analyte isa molecule present on or within a cell, extraction can be performedusing lysing agents including but not limited to denaturing detergentsuch as SDS or non-denaturing detergent such as thesit, sodiumdeoxylate, triton X-100, and tween-20.

In some embodiments, pretreatment can include diluting and/or mixing thesample, and filtering the sample to remove, e.g., red blood cells from ablood sample.

A bodily fluid may be drawn from a patient and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In one embodiment, a lancet punctures the skinand draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. The lancet maybe part of the fluidic device, or part of a reader assembly, or a standalone component. In another embodiment where no active mechanism isrequired, a patient can simply provide a bodily fluid to the fluidicdevice, as for example, could occur with a saliva sample. The collectedfluid can be placed in the sample collection unit within the fluidicdevice. In yet another embodiment, the fluidic device comprises at leastone microneedle which punctures the skin. The microneedle can be usedwith a fluidic device alone, or can puncture the skin after the fluidicdevice is inserted into a reader assembly.

A sample collection unit in a fluidic device may provide a bodily fluidsample from a patient by any of the methods described above. Ifnecessary, the sample may first be processed by diluting the bodilyfluid in a dilution chamber, and/or may be filtered by separating theplasma from the red blood cells in a filtration chamber as describedabove. In some embodiments the sample collection unit, diluting chamber,and filtration chamber may be the same component, and in someembodiments they may be different components, or any two may be the samecomponent and the other may be a separate component. In some embodimentsthere may be more than one sample collection unit in the fluidic deviceor system.

Assay Assembly:

The assay assembly contained in the subject system or fluidic devicecomprises reactants capable of reacting with analytes to yield coloredproducts that are indicative of the presence of the analytes. As usedherein, the term “analytes” refers to any substances in a bodily fluidthat can be used for generating colored products for detection.Exemplary analytes include without limitation drugs, prodrugs,pharmaceutical agents, drug metabolites, a biomarker indicative of adisease, a tissue specific marker, a tissue specific enzyme biomarkerssuch as expressed proteins and cell markers, antibodies, serum proteins,cholesterol, polysaccharides, nucleic acids, gene, protein, or hormone,or any combination thereof. At a molecular level, the analytes can bepolypeptide glycoprotein, polysaccharide, lipid, nucleic acid, and acombination thereof. Preferred detectable analytes include but are notlimited to HDL cholesterol, LDL cholesterol, total cholesterol, lipids,glucose, and enzymes.

As noted above, the assay assembly of the subject system or fluidicdevice is configured to detect analytes based on formation of a coloredproduct from a reaction scheme that is indicative of its presence.Exemplary classes of analytes that can be detected in this mannerinclude: a) analytes that can be converted chemically to a coloredproduct via a color-producing reaction; b) analytes that catalyze theformation of colored products from chemical reactants; and c) analytesthat can be detected through binding of an agent that then participatesin a color-producing reaction, either as a chemical reagent or apromoter of a chemical reaction. Additional examples of analytes thatcan yield colored products are illustrated in e.g., Tietz Textbook ofClinical Chemistry (Second Ed., Burtis and Ashwood, Saunders, 1994).

Analytes that can be converted chemically to a colored product via acolor-producing reaction include enzyme substrates and co-factors.Non-limiting examples of such analytes include glucose, cholesterol, andtriglycerides. In particular, levels of total cholesterol (i.e., the sumof free and esterified cholesterol) in a bodily fluid can bespectrophotometrically measured by well-known color-forming assays byreacting the fluid with reactants including cholesterol esterase,cholesterol oxidase, an oxidizable dye such asn,n-bis(4-sulfobutyl)-3-methylaniline, disodium salt (TODB),4-aminoantipyrine, and horse radish peroxidase.

A vast number of analytes can catalyze the formation of a coloredproducts from chemical reactants, thus are amenable for detection byoptical means. Examples of such analytes include alanineaminotransferase (ALT) and aspartate aminotransferase. Alanineaminotransferase (ALT) is an analyte indicative of liver function. Thereactants for use in this assay may include alphaketoglutarate, pyruvateoxidase, an oxidizable dye such asN,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (TODB),4-aminoantipyrine, and horse radish peroxidase.

The third class of analytes is typically detected via a color-producingimmunoassay, such as an enzyme-linked immunosorbent assay (ELISA). In atypical ELISA, an analyte is specifically bound by an antibody, which inturn is detected by a secondary, enzyme-linked antibody. The linkedenzyme catalyzes a color-producing reaction. Such enzymes include butare not limited to β-galactosidase, alkaline phophatase, and horseradish peroxidase.

The choice of suitable reactants will depend on the particular analytesbeing examined. In general, any reactants capable of reacting withanalytes either directly or indirectly to generate colored products,which can then be detected optically, are suited for use in the subjectsystem. Exemplary reactants include but are not limited to one or moreenzymes, co-factors, dyes, and other reagents as needed to convert theseand analytes to a colored product.

Of particular interest are several color forming reactants for use inthe present invention. In one embodiment, peroxidase reactions arepreferably used to generate colored products. Peroxidase chromogens arewell known in the art, as exemplified by Trinder reagents such as TODBor TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodiumsalt, dehydrate) used in combination with 4-aminoantipyrene, triarylimidazoles, and ABTS (2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonicacid). In the chemistry of peroxidase reactions with Trinder reagents,two colorless organic molecules form a colored product in the presenceof peroxidase and hydrogen peroxide. This peroxidase chemistryadvantageously generates an intensely colored product and is not subjectto interference from substances in blood plasma.

Reactants in the assay assembly can be contained in reaction sites,either as fluids or dry reagents. In the case of dry reagents, thereaction site preferably forms a rigid support on which a reactant canbe immobilized. The reaction site surface is also chosen to providecharacteristics for detection of light absorbance. For instance, thereaction site may be functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, polycarbonate, polypropylene, or combinations thereof.Other appropriate materials may be used in accordance with the presentinvention.

One skilled in the art will appreciate that there are many ways ofimmobilizing various reactants onto a support where reaction can takeplace. The immobilization may be covalent or noncovalent, via a linkermoiety, or tethering them to an immobilized moiety. These methods arewell known in the field of solid phase synthesis and micro-arrays (Beieret al., Nucleic Acids Res. 27:1970-1-977 (1999). Non-limiting exemplarybinding moieties for attaching either nucleic acids or proteinaceousmolecules such as antibodies to a solid support include streptavidin oravidin/biotin linkages, carbamate linkages, ester linkages, amide,thiolester, (N)-functionalized thiourea, functionalized maleimide,amino, disulfide, amide, hydrazone linkages, and among others. Inaddition, a silyl moiety can be attached to a nucleic acid directly to asubstrate such as glass using methods known in the art.

In a preferred embodiment, there are multiple reaction sites in an assayassembly which can allow for detection of multiple analytes of interestfrom the same sample of bodily fluid. In some embodiments there are 2,3, 4, 5, 6, or more reaction sites, or any other number of reactionsites as may be necessary to carry out the intent of the invention.

In embodiments with multiple reaction sites in a fluidic device, eachreaction site may be immobilized with reactants different from reactantsimmobilized at a different reaction site. In a fluidic device with, forexample, three reaction sites, there may be three different reactants,each immobilized to a different reaction site to detect three differentanalytes of interest in the sample.

In some embodiments, the reactants are contained in a reactant chamber.A reactant chamber is preferably in fluid communication with at leastone reaction site, and when the fluidic device is actuated, reactantscontained in the reactant chamber are released into a fluidic channelwithin the fluidic device and introduced into a reaction site. Reactantsmay be contained in reactant chambers as fluids or dry reagents, asdescribed above with respect to reactants contained in reaction sites.In some embodiments there may be two, three, four, five, six, or more,or any number of reactant chambers as are necessary to fulfill thepurposes of the invention.

In addition to color-forming reactants, the present invention mayinclude other reagents. Such reagents can be stored with reactants inreaction sites or reactant chambers, if appropriate. In anotherembodiment reagents are stored separately, and there is at least onereagent chamber. Reagents may be stored in a fluid or dry state, similarto reactants. In some embodiments there may be two, three, four, five,six, or more, or any number of reagent chambers as are necessary tofulfill the purposes of the invention. A reagent chamber is preferablyin fluid communication with at least one reaction site, and when thefluidic device is actuated, reagents contained in said reagent chambersare released into the fluidic channels within the fluidic device andintroducted into a reaction site.

Reagents according to the present invention include without limitationwash buffers, enzyme substrates, dilution buffers, conjugates,enzyme-labeled conjugates, DNA amplifiers, sample diluents, washsolutions, sample pre-treatment reagents including additives such asdetergents, polymers, chelating agents, albumin-binding reagents, enzymeinhibitors, enzymes, anticoagulants, red-cell agglutinating agents,antibodies, or other materials necessary to run an assay in a fluidicdevice. In general, reagents especially those that are relativelyunstable when mixed with liquid are confined in a defined region (e.g. areagent chamber) within the subject fluidic device. The containment ofreagents can be effected by valves that are normally closed and designedfor one-time opening, preferably in a unidirectional manner. In someembodiments the reagents are initially stored dry and liquified uponinitiation of the assay being run on the fluidic device.

In some embodiments a reactant site, reactant chamber or reagent chambercontains approximately about 50 μto about 1 ml of fluid. In someembodiments the chamber may contain about 100 μof fluid. The volume ofliquid in a reactant or reagent chamber may vary depending on the typeof assay being run or the sample of bodily fluid provided.

In preferred embodiments of the invention the fluidic device includes atleast one waste chamber to trap or capture all liquids after they havebeen used in the assay. In preferred embodiments, there is more than onewaste chamber, at least one of which is to be used with a calibrationassembly described herein below. On-board waste chambers also allow thedevice to be easily disposable. The waste chamber is preferably influidic communication with at least one reaction site.

Light Source

A colored product of an analyte-detecting assay of the present inventionis typically detected by measurement of absorbance of light by thecolored product. Light will be directed to the colored product in areaction site from a source that emits a spectrum of light in which atleast one wavelength of light corresponds to the absorption spectrum ofthe colored product. The spectrum of the light emitted by a sourceaccordingly will be similar to the spectrum of the absorbing species inthe colored product of the analyte-detecting reaction. Preferably, theemission spectrum from the light source will overlap the absorptionspectrum of the absorbing species, preferably by at least about 50%,60%, 70%, 80%, 90% or 95%. However, the present invention does notrequire an exact overlap between the light source emission spectrum andthe absorption spectrum of the colored product, as described in theexamples provided herein. Use of monochromatic light sources and/orfilters can generally provide a means to match the characteristics ofthe absorption and the light source.

The colored products detected by the subject system typically have anabsorption range of about 250 nm to about 900 nm. Preferably, the colorto be measured is generally in a visible range of about 400 to about 800nm.

The absorbance of the colored product can be readily detected and in arange that is preferably stoichiometrically or linearly corresponds tothe amount of analyte present. According to Beer's law,absorbance=concentration×extinction coefficient×optical path length.Chromophores in the visible wavelength range and typically used inclinical chemistry have extinction coefficients in the range of about10³-10⁵ L/(mole×cm). As shown in Table 1 of Example 1, a concentrationof 1.5 mM analyte, diluted by 1:30 fold, gives an absorbance of 0.25(44% transmission) when measured at the maximum absorbance (at λ max of500 nm, the extinction coefficient=50,000 L/(mole×cm) with a path lengthof 0.1 cm (typical of single use cartridges). This absorbance is readilymeasurable by simple transmission optical systems.

A variety of light sources may be utilized for the present inventiondepending on the particular type of application and absorbance spectrumrequirements for a given analyte of interest. An example of anappropriate light source includes, but is not limited to, anincandescent bulb, a light emitting diode, luminescent paint, and alaser. Preferably, the light source is an economical, low intensitylight source well suited for point-of-care testing. When coupled with aphotomultiplier tube detector, the number of photons generated by thelight source need only be a few thousand over a measurement interval,which can range from a few milliseconds to a several minutes.

One type of light source applicable for the present invention isluminescent paint. Such paint is generally formulated using very tinyquantities of a long-lived radioisotope together with a material thatglows or scintillates non-destructively when irradiated. The paint canbe appropriately colored by addition of dyes. The paint will generallybe coated on the non-transparent walls of a reaction site where analyteassay chemistry generates a colored product. Light emitted from thepaint can be detected through a transparent surface of the reaction siteto allow measurement of absorbance due to a colored product. Thespectrum of the light emitted will generally be a function of thescintillant material and the absorbance characteristics of the chemistryused in forming a colored product.

Another applicable light source for the present invention is a LightEmitting Diode (LED). A LED can provide colored light at moderateintensity. The spectrum of the emitted light can be selected over thevisible range. A LED typically has a more narrow range of emissionwavelengths of about 30 nm. Thus, use of a LED as a light source willdepend on the absorbance spectrum of an absorbing species used in thedetection of a particular analyte.

Detector

Detection and measurement of colored products generated due to thepresence of a given analyte can be made directly from a reaction site oralternatively from a detection site to which the colored product istransported. Preferably, detection will be made from a reaction site.Unless specified otherwise, the term “reaction site” as used herein willrefer to both the site at which a reaction occurs and at which thecolored product of the reaction is detected. The reaction site willtypically be a well that is cylindrical in shape having a defined lengthbetween two opposed flat surfaces for determination of absorbance. Forexample, the point-of-care fluidic devices of the present inventionmight have a reaction site that is 0.1 cm in length. At least one orboth of the flat surfaces of the reaction site will be transparent toallow detection of the colored product with standard transmissionoptics. The non-transparent surfaces of the reaction site may be made ofopaque, white light scattering material.

The detector of light transmitted from a light source through a reactionsite will be capable of detecting absorbance of light by the coloredproduct in the reaction site. Examples of suitable detectors include,but are not limited to, a photomultiplier tube, a photodiode or anavalanche photodiode.

In a system of the present invention, the position of the light detectorin the system relative to the fluidic device will depend on factors suchas the type of light source used and the relative position of the lightsource to the fluidic device. In the case where the light source is aluminescent paint contained within a reaction site of the device, thedetector will be positioned to detect light emitted from a transparentsurface of the reaction site.

In the situation where the light source is external to a fluidic device,a detector could be positioned either on the same side or an oppositeside of the fluidic device relative to the light source. A reaction sitecan be configured with a single transparent surface, through which lightis both directed to the reaction and detected from the reaction. In thisscenario, a detector is positioned on the same side of the fluidicdevice as the light source, with the detector shielded such that theonly light detected is that from the reaction site of the fluidicdevice. Alternatively, a reaction site can be configured with two flat,opposed transparent surfaces such that the reaction site is effectivelyan optical cuvette. In this configuration, the light source would emitlight to one side of the reaction site in the fluidic device and thedetector would detect the light transmitted through the colored productto the opposite side of the reaction site in the fluidic device.

The fluidic devices of the present invention preferably function ashandheld devices in a point-of-care system. The term “handheld” refersto a device that is both small and light enough to be easily held in anadult's hand, and can readily be placed by hand into operation within apoint-of-care system. A handheld device of the present invention mayassume a variety of overall configurations, such as rectangular,triangular, circular, oval and so forth. Regardless of the overallconfiguration, a handheld device of the present invention may typicallybe enclosed within rectangular dimensions of about 30×30×15 cm(length×width×height), or about 12×10×5 cm, or about 8×6×1.5 cm, andeven smaller, such as about 7×5×1 cm.

A “point-of-care” system as used herein refers to a system that may beused at a patient's home, bedside, or other environment for performingany type of bodily fluid analysis or test outside of a centrallaboratory. A point-of-care system of the present invention will enabletesting to be efficiently carried out by a patient or an assistant, ahealth care provider, and so forth. A point-of-care system preferablyhas dimensions and a configuration that allows it to be convenientlytransported to a user's desired environment and readily used fortesting.

FIG. 1 illustrates an exemplary system of the present invention. Asillustrated, a fluidic device provides a bodily fluid from a patient andcan be inserted into a reader assembly. The fluidic device may take avariety of configurations and in some embodiments the fluidic device maybe in the form of a cartridge. An identifier (ID) detector may detect anidentifier on the fluidic device. The identifier detector communicateswith a communication assembly via a controller which transmits theidentifier to an external device. Where desired, the external devicesends a protocol stored on the external device to the communicationassembly based on the identifier. The protocol to be run on the fluidicdevice may comprise instructions to the controller of the readerassembly to perform the protocol on the fluidic device, including butnot limited to a particular assay to be run and a detection method to beperformed. Once the assay is performed on the fluidic device, a signalindicative of an analyte in the bodily fluid sample is generated anddetected by a detector. The detected signal may then be communicated tothe communications assembly, where it can be transmitted to the externaldevice for processing, including without limitation, calculation of theanalyte concentration in the sample.

FIG. 2 illustrates exemplary layers of a fluidic device according to thepresent invention prior to assembly of the fluidic device. FIGS. 3 and 4show a top and bottom view, respectively, of an exemplary fluidic deviceafter the device has been assembled. The different layers are designedand assembled to form a three dimensional fluidic channel network. Asample collection unit provides a sample of bodily fluid from a patient.A reader assembly comprises actuating elements (not shown) that canactuate the fluidic device to start and direct the flow of a bodilyfluid sample and assay reagents in the fluidic device. In someembodiments actuating elements first cause the flow of sample in thefluidic device from a sample collection unit 4 to reaction sites 6, andthen to waste chamber 8 following completion of reactions in the sites.If necessary for a given reaction, the actuating elements initiate flowof reagents from reagent chambers 10 to reaction sites, and then towaste chamber 8 in a manner similar to that of the sample.

A fluidic device of the present system can run a variety of assays,regardless of the analyte being detected from a bodily fluid sample. Aprotocol dependent on the identity of the fluidic device may betransferred from an external device where it can be stored to a readerassembly to enable the reader assembly to carry out the specificprotocol on the fluidic device. In preferred embodiments, the fluidicdevice has an identifier (ID) that is detected or read by an identifierdetector. The identifier can then be communicated to a communicationassembly, where it can then be transferred or transmitted to an externaldevice.

In one embodiment, a bodily fluid sample is provided to a fluidicdevice, which is then inserted into a reader assembly. In someembodiments the fluidic device is partially inserted manually, and thena mechanical switch in the reader assembly automatically properlypositions the fluidic device inside the reader assembly. Any othermechanism known in the art for inserting a disk or cartridge into adevice may be used as well. In some embodiments only manual insertionmay be required.

In preferred embodiments the reader assembly houses a controller whichcontrols a pump and a series of valves to control and direct the flow ofliquid within the fluidic device. In some embodiments the readerassembly may comprises multiple pumps. The sample and reagents arepreferably pulled through the fluidic channels by a vacuum force createdby sequentially opening and closing at least one valve while activatinga pump within the reader assembly. Methods of using at least one valveand at least one pump to create a vacuum force are well known. While anegative pulling force may be used, a positive pushing force may also begenerated by at least one pump and valve according to the presentinvention. In other embodiments movement of fluid on the fluidic devicemay be by electro-osmotic, capillary, piezoelectric, or microactuatoraction.

One of the advantages of the present invention is that any reagentsnecessary to perform an assay on a fluidic device according to thepresent invention are preferably on-board, or housed within the fluidicdevice before, during, and after the assay. In this way the only inletor outlet from the fluidic device is preferably the bodily fluid sampleinitially provided by the fluidic device. This design also helps createan easily disposable fluidic device where all fluids or liquids remainin the device. The on-board design also prevents leakage from thefluidic device into the reader assembly which should remain free fromcontamination from the fluidic device.

Method of Use

The subject apparatus and systems provide an effective means for highthroughput and/or real-time detection of analytes present in a bodilyfluid from a subject. The detection methods may be used in a widevariety of circumstances including identification and quantification ofanalytes that are associated with specific biological processes,physiological conditions, disorders or stages of disorders. As such, thesubject apparatus and systems have a broad spectrum of utility in, e.g.drug screening, disease diagnosis, phylogenetic classification, parentaland forensic identification. The subject apparatus and systems are alsoparticularly useful for advancing preclinical and clinical stagedevelopment of therapeutics, improving patient compliance, monitoringadverse drug responses associated with a prescribed drug, and developingindividualized medicine.

Accordingly, in one embodiment, the present invention provides a methodof detecting an analyte in a bodily fluid from a subject. The methodtypically involves the steps of (a) introducing a sample of bodily fluidinto a fluidic device having a sample collection unit and an assayassembly, the assay assembly having reactants that are capable ofreacting with an analyte; (b) allowing the sample of bodily fluid toreact with the reactants contained within the assay assembly to yield acolored product having an absorbance spectrum corresponding to at leastone wavelength from a light source; (c) transmitting light having the atleast one wavelength to the fluidic device from the light source; and(d) detecting absorption of light of the at least one wavelengthtransmitted to the fluidic device, wherein the absorption is indicativeof the presence of the analyte in said bodily fluid.

Any bodily fluids suspected to contain an analyte of interest can beused in conjunction with the subject methods of detection. Commonlyemployed bodily fluids include but are not limited to blood, serum,saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginalfluid, interstitial fluids derived from tumorous tissue, andcerebrospinal fluid. The volume of bodily fluid to be used in methods ofthe present invention is generally less than about 500 microliters, andpreferably less than about 50 microliters. Where desired, a sample of 1to 50 microliters, 1 to 40 microliters, 1 to 30 microliters, 1 to 20microliters, or 1 to 10 microliters can be used for detecting an analyteusing the subject fluidic device.

A bodily fluid may be drawn from a patient and brought into the fluidicdevice in a variety of ways, including but not limited to, lancing,injection, or pipetting. In one embodiment, a lancet punctures the skinand draws the sample into the fluidic device using, for example,gravity, capillary action, aspiration, or vacuum force. The lancet maybe part of the fluidic device, or part of a reader assembly, or as astand alone component. Where needed, the lancet may be activated by avariety of mechanical, electrical, electromechanical, or any other knownactivation mechanism or any combination of such methods. In anotherembodiment where no active mechanism is required, a patient can simplyprovide a bodily fluid to the fluidic device, as for example, couldoccur with a saliva sample. The collected fluid can be placed in thesample collection unit within the fluidic device. In yet anotherembodiment, the fluidic device comprises at least one microneedle whichpunctures the skin. The microneedle can be used with a fluidic devicealone, or can puncture the skin after the fluidic device is insertedinto a reader assembly.

In some embodiments a microneedle is about the size of a human hair andhas an integrated microreservoir or cuvette. The microneedle maypainlessly penetrate the skin and draw a small blood sample. Morepreferably, the microneedle collects about 0.01 to about 1 microliter,preferably about 0.05 to about 0.5 microliters and more preferably about0.1 to about 0.3 microliters of capillary blood. In some embodiments amicroneedle may be constructed out of silicon and is about 10 to about200 microns in diameter, preferably about 50 to about 150 microns indiameter, and most preferably about 100 microns in diameter, makingtheir application to the skin virtually painless. To ensure that acapillary is actually struck by a needle, a plurality of microneedlesmay be used for sample collection. Such microneedles may be of the typemarketed by Pelikan (Palo Alto, Calif.) and/or Kumetrix (Union City,Calif.). U.S. Pat. No. 6,503,231 discloses microneedles which may beused with the present invention.

In preferred embodiments a microneedle is only used once and thendiscarded. In some embodiments a mechanical actuator can insert andwithdraw the microneedle from the patient, discard the used needle, andreload a new microneedle.

In some embodiments the method of detecting an analyte in a bodily fluidfrom a subject includes metering a predetermined portion of the sample,in which this predetermined portion is assayed for the presence ofanalytes. The volume of the predetermined portion will preferably beless than about 500 microliters, more preferably about less than 50microliters, or even more preferably, the volume is about 10microliters.

A precise sample volume is determined by several features. In oneembodiment a subject places a sample of bodily fluid into the samplecollection well, after which the sample is drawn into a metering channelby capillary action until it reaches a stop junction at the entrance ofthe mixing chamber. The metering channel preferably has physicaldimensions and surface characteristics which reliably promote flow ofblood from the sample collection well.

In a preferred embodiment, a predetermined portion of a sample isdiluted and mixed with a diluent to yield a diluted sample, which isthen assayed for the presence of analytes. A predetermined portion isdiluted with a diluent that is typically contained in a diluent chamber,with the portion and diluent being mixed in a mixing chamber.Preferably, the diluent is flowed into the metering channel, whichflushes the sample into the mixing chamber. A precise volume of diluentis stored in the dilution chamber. A precise volume of diluent, aprecise volume of the predetermined portion of a sample, and efficientcombination and mixing of the two volumes allows the sample to bediluted with a high degree of precision.

In some embodiments, the fluid sample will be filtered before entering areaction chamber. For example, blood may be filtered to remove red bloodcells. Where a sample is diluted before assaying, filtering willtypically occur after dilution. Filtering will occur in a filterchamber, through which the sample is transported before entering into areaction site.

A variety of assays may be performed on a fluidic device according tothe present invention to detect an analyte of interest in a sample.Analytes that may be detected by the subject methods include, but arenot limited to, drugs, drug metabolites, biomarkers indicative ofdisease, tissue specific markers, tissue specific enzymes, hormones,antibodies, pathogens, HDL cholesterol, LDL cholesterol, totalcholesterol, lipids, and glucose.

The subject methods involve reactants that are capable of reacting withan analyte of interest to generate a color product for detection byoptical means. The choice of reactants will depend on the particularanalyte being examined.

For detection of levels of total cholesterol (i.e., the sum of free andesterified cholesterol) in a bodily fluid, reactants includingcholesterol esterase, cholesterol oxidase, an oxidizable dye such asn,n-bis(4-sulfobutyl)-3-methylaniline, disodium salt (TODB),4-aminoantipyrine, and horse radish peroxidase can be employed. In thisreaction scheme, cholesterol esterase converts esterified cholesterol tofree cholesterol. Cholesterol oxidase transforms the free cholesterolinto cholest-4-ene-3-one and hydrogen peroxide. The amount of hydrogenperoxide generated can be quantified by a spectrophotometric assay, forexample the oxidative coupling of 4-aminoantipyrine and TODB in thepresence of peroxidase to form a chromophore. The amount of chromophoreformed is then measured by light attenuation (absorbance), whichcorresponds to the amount of total cholesterol.

Measuring the ALT levels as a way for assaying liver function can becarried out by reacting the analyte from a bodily fluid with reactantssuch as alphaketoglutarate, pyruvate oxidase, an oxidizable dye such asN,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt (TODB),4-aminoantipyrine, and horse radish peroxidase. In this assay reactionscheme, ALT catalyzes the transfer of amino groups from L-alanine toalphaketoglutarate, producing pyruvate and glutamate. Pyruvate oxidaseoxidizes the pyruvate to acetylphosphate and hydrogen peroxide.Horseradish peroxidase catalyzes the reaction of the peroxide reactswith TODB to form a colored product at a rate proportional to the ALTconcentration of the sample. The resultant colored product in thereaction is measured by light absorbance.

The methods of the present invention can also be used to detectanalytes, such as small molecule drugs, biomarkers, hormones, andantibodies, through binding of an agent that then participates in acolor-producing reaction. For example, an analyte can be detectedthrough binding and color formation that occurs in immunoassays, such asan enzyme-linked immunosorbent assay (ELISA). In a typical ELISA, ananalyte is specifically bound by an antibody, which in turn is detectedby a secondary, enzyme-linked antibody. The linked enzyme catalyzes acolor-producing reaction. Enzymes such as β-galactosidase, alkalinephophatase and horse radish peroxidase are often utilized for colorformation in ELISAs. The light absorbance of colored products generatedin an ELISA is typically in a range well suited for the presentinvention. Reactants of the present invention accordingly will includereagents for an ELISA or similar immunoassay. Unlike typical assays fordetection of analytes that are chemical reactants or promote a chemicalreaction, ELISA immunoassays require wash steps, and thus generally willoccur in separate, dedicated reaction sites.

A colored product will be detected in methods of the present inventionthrough measurement of absorbance of light by the colored product. Thelight to be transmitted in the methods of the present invention will befrom a source that emits a spectrum of light in which at least onewavelength of light corresponds to the absorption spectrum of thecolored product. The range of absorption spectra of colored productswill correspond to a wavelength range of about 250 nm to about 900 nm.Preferably, the color to be measured is generally in a visible range ofabout 400 to about 800 nm. The spectrum of light emitted by a sourceaccordingly will be similar to the absorption spectrum of the coloredproduct. Preferably, the emission spectrum from a light source willexactly overlap the absorption spectrum of the absorbing species.However, an exact overlap between the light source emission spectrum andthe absorption spectrum of the colored product is not required formeasurement by the methods of the present invention, as described in theexamples provided herein. Monochromatic light sources and/or filtersgenerally can be used to provide a means to match the characteristics ofthe absorption and the light source.

A variety of light sources may be utilized for the present inventiondepending on the particular type of application and absorbance spectrumrequirements for a given analyte of interest. An example of anappropriate light source includes, but is not limited to, anincandescent bulb, a light emitting diode, luminescent paint, and alaser.

The position of the light source relative to the reaction site willdepend on the particular source of light. Typically, a light source willtransmit light into the reaction site through a transparent, flatsurface of the reaction site. In this scenario, the light source will beexternal to the fluidic device, with the reaction site aligned with thelight source so that light is transmitted directly into the reactionsite. To enable measurement from several reaction sites, the fluidicdevice and light source will be moveable relative to each other to allowalignment of more than one individual reaction site with the lightsource. Either the fluidic device, light source, or a combination of thetwo can be moveable within a system to allow alignment.

As an alternative to light being transmitted into the reaction site froman external source, the methods of the present invention can utilize aluminescent paint as an internal light source. In this scenario, theluminescent paint will emit light through a colored product contained inthe reaction site. For example, the reaction site could have acylindrical shape, with two flat opposed surfaces, with one beingtransparent, the other being coated with a luminescent paint. Theluminescent paint will emit light through the colored product, whichcould be detected by a detector as detailed below. Luminescent paint isgenerally formulated using very tiny quantities of a long-livedradioisotope together with a material that glows or scintillatesnon-destructively when irradiated. The paint can be appropriatelycolored by addition of dyes. The spectrum of light emitted willgenerally be a function of the scintillant material and the absorbancecharacteristics of the chemistry used in forming a colored product.

The light generated in the methods of the present invention will bedetected by a detector that will be external to the fluidic device.Examples of suitable detectors include, but are not limited to, aphotomultiplier tube, a photodiode or an avalanche photodiode.

The position of the light detector relative to the fluidic device willdepend on the light source used and its relative position to the fluidicdevice. In the case where the light source is a luminescent paintcontained within a reaction site of the device, the detector can bepositioned as necessary to be aligned with a transparent surface of thereaction site to detect light emitted through a colored product.

In the situation where the light source is external to a fluidic device,a detector could be positioned either on the same side or an oppositeside of the fluidic device relative to the light source. A reaction sitecan be configured with a single transparent surface to allow both lighttransmission into the site and detection from the site. In thisscenario, a detector would be positioned on the same side of the fluidicdevice as the light source, and shielded such that the only lightdetected is that emitted from the reaction site of the fluidic device.Alternatively, a reaction site can be configured with two flat, opposedtransparent surfaces such that the reaction site is effectively anoptical cuvette. In this configuration, the light source would transmitlight to one side of the reaction site in the fluidic device and thedetector would detect the light transmitted through the colored productto the opposite side of the reaction site in the fluidic device. Ineither scenario, the detector will be positioned to align with thereaction site to detect light emission.

To allow measurement from several reaction sites, the fluidic device andlight detector will be moveable relative to each other to allowalignment of more than one individual reaction site with the lightdetector. Either the fluidic device, the detector, or a combination ofthe two can be moveable to allow alignment.

In addition to detection of the presence of an analyte in a bodilyfluid, the methods of the present invention also provide forquantitation of the concentration of an analyte in a bodily fluidthrough measurement of absorbance. Concentration of the analyte isrelated to the amount of light adsorbed by the colored product. In thecase of analytes that can be converted directly or indirectly intocolored product, such as cholesterol, the conversion to product istypically stoichiometric. For instance, the amount of color produced canlinearly increase with the amount of analyte present. The correspondingabsorbance can be proportionately related to the amount of colorproduced, and therefore the concentration of analyte present. However,at high concentrations, the proportionality of absorbance toconcentration set forth by Beer's Law does not necessarily hold. Thus,an accurate measurement of analyte present at high concentration maydepend on an appropriate dilution of a bodily fluid, the characteristicsof the particular absorbing species, and the length of cell path fromwhich determination of absorbance is made.

Analytes that are detected by their ability to catalyze formation of acolored product, such as the enzyme ALT, can be quantified following aparticular length of reaction time. By allowing an analyte enzyme toreact for a fixed period of time, appropriate quantities of measurable,colored product can be generated. For example, a fixed period of timeunder conditions in which the amount of an analyte enzyme is arate-limiting factor can give rise to uM-nM quantities of coloredproduct. The quantity of product generated can be measured at the end ofthe time period by measurement of light attenuation and determination ofabsorbance. The amount of analyte can then be determined based on theamount of product generated over time, based on known kinetics of agiven analyte under the conditions of a particular assay used. Anaccurate measurement of analyte will depend on the particular analytebeing examined, conditions under which it is assayed (at what dilution,temperature, and so on), the characteristics of the particular absorbingspecies, and the length of cell path from which determination ofabsorbance is made.

In some embodiments immunoassays are run on the fluidic device. Whilecompetitive binding assays, which are well known in the art, may be runin some embodiments, in preferred embodiments a two-step method is usedwhich eliminates the need to mix a conjugate and a sample beforeexposing the mixture to an antibody, which may be desirable when verysmall volumes of sample and conjugate are used, as in the fluidic deviceof the present invention. A two-step assay has additional advantagesover the competitive binding assays when use with a fluidic device asdescribed herein. It combines the ease of use and high sensitivity of asandwich (competitive binding) immunoassay with the ability to assaysmall molecules.

In an exemplary two-step assay, the sample containing analyte firstflows over a reaction site containing antibodies. The antibodies bindthe analyte present in the sample. After the sample passes over thesurface, a solution with analyte conjugated to a marker at a highconcentration is passed over the surface. The conjugate saturates any ofthe antibodies that have not yet bound the analyte. Before equilibriumis reached and any displacement of pre-bound unlabelled analyte occurs,the high-concentration conjugate solution is washed off. The amount ofconjugate bound to the surface is then measured by the appropriatetechnique, and the detected conjugate is inversely proportional to theamount of analyte present in the sample.

The methods of the present invention provide for monitoring more thanone pharmacological parameter useful for assessing efficacy and/ortoxicity of a therapeutic agent. For the purposes of this invention, a“therapeutic agent” is intended to include any substances that havetherapeutic utility and/or potential. Such substances include but arenot limited to biological or chemical compounds such as simple orcomplex organic or inorganic molecules, peptides, proteins, orpolynucleotides. A vast array of compounds can be synthesized, forexample polymers, such as polypeptides and polynucleotides, andsynthetic organic compounds based on various core structures, and theseare also included in the term “therapeutic agent”. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like. It should be understood, although notalways explicitly stated that the agent is used alone or in combinationwith another agent, having the same or different biological activity asthe agents identified by the inventive screen. The agents and methodsalso are intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters in real time from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the patient. Similarly, if any of the pharmacodynamic (PD)parameters fall outside the desired range during a treatment regime,immediate action may have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to store profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore patients to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the device for improved accuracy indetermining drug pathways and efficacy in cancer studies.

Being able to monitoring the rate of change of an analyte concentrationor PD or PK over a period of time in a single subject, or performingtrend analysis on the concentration, PD, or PK, whether they areconcentrations of drugs or their metabolites, can help preventpotentially dangerous situations. For example, if glucose were theanalyte of interest, the concentration of glucose in a sample at a giventime as well as the rate of change of the glucose concentration over agiven period of time could be highly useful in predicting and avoiding,for example, hypoglycemic events. Such trend analysis has widespreadbeneficial implications in drug dosing regimen. When multiple drugs andtheir metabolites are concerned, the ability to spot a trend and takeproactive measures is often desirable.

The present invention allows for automatic quantification of apharmacological parameter of a patient as well as automatic comparisonof the parameter with, for example, the patient's medical records whichmay include a history of the monitored parameter, or medical records ofanother group of subjects. Coupling real-time analyte monitoring with anexternal device which can store data as well as perform any type of dataprocessing or algorithm, for example, provides a device that can assistwith typical patient care which can include, for example, comparingcurrent patient data with past patient data.

Where a statistically significant discrepancy exists between thedetected values and the threshold value, a further action may be takenby a medical practitioner. Such action may involve a medical action suchas adjusting dosage of the therapeutic agent; it may also involve abusiness decision such as continuing, modifying, or terminating theclinical trial.

One advantage of the current invention is that assay results can besubstantially immediately communicated to any third party that maybenefit from obtaining the results. For example, once the analyteconcentration is determined at the external device, it can betransmitted to a patient or medical personnel who may need to takefurther action. The communication step to a third party can be performedwirelessly, and by transmitting the data to a third party's hand helddevice, the third party can be notified of the assay results virtuallyanytime and anywhere. Thus, in a time-sensitive scenario, a patient maybe contacted immediately anywhere if urgent medical action may berequired.

In some embodiments a patient may be provided with a plurality offluidic devices to use to detect a variety of analytes. A subject may,for example, use different fluidic devices on different days of theweek.

In some embodiments, the methods of the present invention are applicablefor obtaining pharmacological data useful for assessing efficacy and/ortoxicity of a pharmaceutical agent from a test animal. When usinglaboratory animals in preclinical testing of a pharmaceutical agent, itis often necessary to kill the test subject to extract enough blood toperform an assay to detect an analyte of interest. This has bothfinancial and ethical implications, and as such it may be advantageousto be able to draw an amount of blood from a test animal such that theanimal does not need to be killed. In addition, this can also allow thesame test animal to be tested with multiple pharmaceutical agents atdifferent times, thus allowing for a more effective preclinical trial.On average, the total blood volume in a mouse, for example, is 6-8 mL ofblood per 100 gram of body weight. A benefit of the current invention isthat only a very small volume of blood is required to performpreclinical trials on mice or other small labaratory animals. In someembodiment between about 1 microliter and about 50 microliters aredrawn. In preferred embodiment between about 1 microliter and 10microliters are drawn. In preferred embodiments about 5 microliters ofblood are drawn.

A further advantage of keeping the test animal alive is evident in apreclinical time course study. When multiple mice, for example, are usedto monitor the levels of an analyte in a test subject's bodily fluidover time, the added variable of using multiple subjects is introducedinto the trial. When, however, a single test animal can be used as itsown control over a course of time, a more accurate and beneficialpreclinical trial can be performed.

In some embodiments the methods of the present invention can be used inmethods of automatically monitoring patient compliance with a medicaltreatment. After determination of an analyte in a bodily fluid, thelevel of analyte can be compared with a known profile associated withthe medical treatment to determine if the patient is compliant ornoncompliant with the medical treatment; and notifying a patient of thecompliance or noncompliance.

Noncompliance with a medical treatment, including a clinical trial, canseriously undermine the efficacy of the treatment or trial. As such, insome embodiments the system of the present invention can be used tomonitor patient compliance and notify the patient or other medicalpersonnel of such noncompliance. For example, a patient taking apharmaceutical agent as part of medical treatment plan can take a bodilyfluid sample which is assayed as described herein, but a detectedmetabolite concentration, for example, may be at an elevated levelcompared to a known profile thereby indicating multiple doses of thepharamaceutical agent have been taken. Such a known profile may belocated or stored on an external device.

The following examples illustrate and explain the invention. The scopeof the invention is not limited by these examples.

EXAMPLES Example 1

Trinder Reagent Spectrum

Several color forming chemistries are applicable for use in the presentinvention, including those of peroxidase reactions. Peroxidasechromophores are well known in the art, as exemplified by Trinderreagents such as TODB or TOOS. A Trinder reagent will generate areaction product having an absorption spectrum such as that exemplifiedin FIG. 8. As shown in FIG. 8, the width of the absorption spectrum atabout half height of spectrum is about 100 nm. The width of the spectrumindicates that the absorption characteristics of a Trinder reagent makemeasurement of absorption applicable over a range of wavelengths.

Example 2

Assay Simulation for a Light Source and Absorbing Species HavingMatching Spectrums

In the present invention, a light source may have an emission spectrumthat perfectly overlaps with the absorption spectrum of the absorbingspecies. Using values that are typical for the chemistry and devices ofthe present invention, Table 1 shows the calculation for one analyteconcentration. As shown in Table 1, an analyte having a concentration of1.5 mM gives an absorbance of 0.25 (44% transmission) after dilution1:30 when measured at the maximum absorbance (at λ max=50,000) with apathlength of 0.1 cm, which would be typical of single use cartridges.FIG. 9 demonstrates the spectral response at this concentration, fromwhich it can be seen that the best response is at λ max of 500 nm.

TABLE 1 Conditions for spectra Luminescence λmax 500 nm Half width 30 nmIntensity 100000 counts (total) Absorption λmax 500 nm Half width 40 nmεM (λmax) 50000 Pathlength, l 0.1 cm Conc. 1.50E−03M (sample) Dilution30 Fold A @ λmax 2.50E−01    ΔT@ λmax 4.38E+01%

Example 3

Cholesterol Assay

Using the parameters given in Example 2, the response of a cholesterolassay is shown in FIG. 10 based on attenuation of light at λ max. Asshown in FIG. 10, the assay signal measured at λ max is well modulatedover the clinical range of tested cholesterol levels.

Example 4

Assay Simulation for a Light Source and Absorbing Species Having OffsetSpectrums

In this example, the parameters are as given in Example 2, with theexception that there is a large offset between the spectrum of the lightsource and the absorption spectrum, with λ max being 50 nm higher forthe absorption spectrum (550 nm rather than 500 nm). As seen in FIG. 11,the light attenuation at the λ max of emission (500 nm) is much lessthan for the ideal case, as shown in FIG. 9. At higher wavelengths,however, the fractional signal modulation between the two spectrums isimproved, albeit at a lower signal level of absorption than that seen atλ max. While the light emission and absorption spectrums will preferablyoverlap exactly, the overlap need not be an exact match for utility inthe present invention.

Example 5

Use of an Edge Filter to Improve Signal Modulation

As shown in Example 4, overlap between light emission and absorptionspectrums need not be an exact match for use in the present invention.However, monochromatic light sources and/or filters can generally beused to create a near exact match of the characteristics between a lightsource and the colored product absorption. Using the same parameters asused in Example 4, FIG. 12 demonstrates use of an edge filter with acut-off of 490 nm used on either the light transmission or detectionside of the optical system to improve signal modulation.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of detecting an analyte in asmall-volume blood sample obtained from a subject, comprising: a)obtaining a sample of blood from a subject by lancing or pipetting, saidblood sample having a volume of less than about 500 μL; b) pre-treatingthe blood sample by one or more of diluting, mixing, drying,evaporating, centrifuging, and extracting; c) introducing the bloodsample into a fluidic device comprising a sample collection unit and anassay assembly, said assay assembly comprising reactants that arecapable of reacting with said analyte; d) allowing the blood sample toreact with said reactants contained within said assay assembly to yielda colored product having an absorbance spectrum corresponding to atleast one wavelength from a light source located within said fluidicdevice; e) transmitting the at least one wavelength to the fluidicdevice from said light source, wherein said light source comprises aluminescent paint; and f) detecting a change in absorption of light ofthe at least one wavelength transmitted to the fluidic device, whereinsaid change in absorption is indicative of the presence of the analytein the blood sample.
 2. The method of claim 1, wherein the amount ofabsorption is related to the analyte concentration in the blood sample.3. The method of claim 1, wherein the amount of absorption isstoichiometrically related to the analyte concentration in the bloodsample.
 4. The method of claim 1, wherein the light source furthercomprises a light emitting diode.
 5. The method of claim 1, wherein saidluminescent paint is coated in said assay assembly.
 6. The method ofclaim 1, wherein the analyte is selected from the group consisting ofdrugs, drug metabolites, biomarkers indicative of a disease, tissuespecific markers, and tissue specific enzymes.
 7. The method of claim 1,wherein the analyte is selected from the group consisting of HDLcholesterol, LDL cholesterol, total cholesterol, lipids, glucose, andenzymes.
 8. The method of claim 1, wherein the wavelength is in a rangeof about 400 to about 800 nm.
 9. The method of claim 1, wherein saidfluidic device detects a plurality of analytes and said fluidic devicecomprises reactants for said plurality of analytes.
 10. The method ofclaim 1, wherein said assay assembly is configured to i) run anenzymatic assay yielding a colored product, or ii) run an immunoassay,or iii) both i) and ii).
 11. The method of claim 1, wherein thereactants are selected from the group of enzymes, enzyme substrates, andcombinations thereof.
 12. The method of claim 1, wherein said sample ofbodily fluid is less than about 50 ul.
 13. The method of claim 1,further comprising the step of quantifying the amount of said analytepresent in said bodily fluid after said detecting step.
 14. The methodof claim 1, wherein said introducing comprises metering a predeterminedportion of said sample to be assayed in said sample collection unit,wherein said predetermined portion of the sample consists of a portionof the sample having a volume determined by the dimensions of an elementof said sample collection unit.
 15. The method of claim 14, furthercomprising diluting and mixing said predetermined portion of said samplewith a diluent in said fluidic device after the metering step to yield adiluted sample.
 16. The method of claim 15, wherein said predeterminedportion of said sample is less than 50 ul.
 17. The method of claim 15,wherein said predetermined portion of said sample is less than 20 ul.18. The method of claim 15, wherein said predetermined portion of saidsample is about 10 ul.
 19. The method of claim 15, further comprisingfiltering said diluted sample before allowing said diluted sample toreact with said reactants.